It has been proposed heretofore to bioassay the sensitivity of tumor cells removed surgically from humans or animals by exposing cultures of such cells to varying dosages of therapeutic agents such as drugs, or radiation by x-rays, gamma rays and the like. Such assay permits prescription of effective, but non-lethal, doses of therapeutic agents for the specific destruction of particular tumor cells, as represented by specimen cells removed from a tumor bearing host. In general, such procedures have been difficult and expensive to perform because of simultaneous growth of more prolific normal cells, including fibroblasts, with the tumor cells where attachment surfaces are available. In general, fibroblasts readily grow in vitro, frequently as the predominate cell type in a mixture of normal and tumor cells to be assayed.
Several procedures are known for isolating particular portions of tumor cells from normal cells. One particular procedure is disclosed in U.S. Pat. No. 4,411,990 issued Oct. 25, 1983 to Salmon, et al. It discloses a technique for an in vitro bioassay of human tumors by growing tumor stem-cells from explanted tumors in animals and humans, or other tumor cell lines. Tumor stem cell colonies are then propagated by plating or gelling them in a carrier nutrient medium in a permeable agar overlayer. The layer of tumor stem-cell colonies overlies a similar permeable layer containing a gelled feeder nutrient medium. The two gelled layers of the system are semi-solid, or solid, with the underlying layer serving to maintain separation of the cells from each other and from the surface of the culture dish, while providing the tumor stem-cells with the requisite feeder nutrients and growth factors. In general, the number of tumor stem-cells is within a given range, and after incubation or growth during a prescribed period of time under conventional incubation temperatures and humidity, the number of viable colony forming tumor stem-cells is counted. The cells are subjected to chemical or radiation therapy at desired doses as a measure of the specificity of such therapy for destruction of the explanted tumor stem-cells during incubation.
While the foregoing method has found application to numerous varieties of tumors, the method presents particular problems. First, it requires the careful preparation of a single cell suspension, carefully preparing both the nutrient solution and plating solution, dispersing the tumor stem cell colonies in the solidified gel and then identifying tumor stem cell colonies in the explanted cell population. The number of viable tumor stem cell colonies are counted at the end of the test and numerically analyzed. Additionally, the method is quite specific to the subjective enumeration of tumor stem cell colonies rather than tumor cells generally. Because tumor cell preparations from surgically removed tumor tissue are contaminated by normal cells including fibroblasts, and because most growth conditions are less than optimal for the tumor cells, prior known methods for growth of tumor cells within a semisolid matrix have been both erratic and unsatisfactory. Accordingly, the ability to provide a very favorable growth environment for the tumor cells to the exclusion of normal cells is made possible by practice of the present invention. Using highly permeable semisolid growth chambers having an enclosed annular volume with continuous feeding and dilution of waste products substantially simplifies such suspension culture.
It has also been known heretofore to cultivate cells and cell lines in vitro, as in glass or plastic flasks or test tubes. Such procedures are particularly suited for growth of attachment-dependent cells, including fibroblasts, which must attach to a solid substratum in order to proliferate. However in general, such growth from surgically removed tumor tissue includes attachment of both anchorage-dependent and anchorage-independent cells to the surface of the flask or test tube and results in a culture containing both normal and tumor cells.
Various methods of supporting cell growth in nutrient media have been disclosed in the prior art. Further, various surfaces have been provided which favor growth of certain cells by favoring specific growth characteristics, such as anchorage dependence.
Using prior known techniques, it has been difficult to establish various kinds of cell lines which are based on human tumor cells. In general, the success rate in establishing such growth from explanted cells of some human tumors, for example, may be as low as from 1 to 10 percent. Further, it has long been desirable to establish homogenous cell lines of indefinite life as sources of desirable biological products generated by their growth for manufacture of vaccines, antibodies, hormones, etc. Heretofore, interference by normal cells has made specific growth of some tumor cells, or clones of other cells having indefinite life, particularly difficult.
Methods of establishing current cell lines heretofore have not been satisfactory since the life span of normal cells tends to be relatively short in that they are finite; that is, the number of replications before they die appears to be genetically preset. Presently, tumor cell lines are known that are at least thirty years old, but evidence no biological change or deterioration of the growth characteristics of the cell line. Accordingly, cancer or tumor cells appear to replicate indefinitely. This permits such cells, including hybridomas or monoclonal antibodies formed therefrom (based on cell hybridization technology), to be used for long-term production of biologically useful molecules. It also makes possible measurement of the immune response of cells infected by viruses. Also, where antigens activate such cells, they may be used to develop antibodies capable of inactivating molecules such as allergens in foods or air, as well as tumors and viruses.